Enzymatic process for the synthesis of organo-fluorine compounds

ABSTRACT

There is described a process for the synthesis of a fluoronucleoside compound, said process comprising mixing a substrate and an enzyme from  Streptomyces cattelya  as catalyst. The process may be used to produce an  18 F labelled fluoronucleoside compound. There is also described an enzyme derived from  Streptomyces cattelya  which has the capacity to catalyse the synthesis of a fluoronucleoside compound.

The present invention relates to a process for the enzymatic synthesisof organo-fluorine compounds, and to an enzyme having the capacity tomediate carbon-fluorine bonds.

Organo-fluorine compounds are hugely important in the fine chemicals,agrochemicals and pharmaceutical industries (blockbuster drugs include5-fluorouracil, Prozac, meflaquin, and there are hundreds of othercommercial entities) Organo-fluorine compounds also have a significantimpact in the materials industry (ferromagnetic materials, Teflon etc).6.6% of all of the compounds registered in Chemical Abstracts (there aremore than 10 million) contain a fluorine atom. There is consequently agreat deal of commercial interest in organo-fluorine compounds. Allorgano-fluorine compounds are necessarily prepared using noxious andhighly toxic fluorinating reagents of anthropogenic origin. Most methodsof fluorination involve toxic and environmentally incompatibleprocesses. Additionally the chemical selectivity of processes tosynthesise organo-fluorine compounds is often low. There are currentlyno biotechnological routes for introducing fluorine into organiccompounds. To date the enzymatic synthesis of organo-fluorine compoundshas been an elusive goal.

Two scientific papers (J. Am. Chem. Soc., 2001, 123, 4350 and AngewChem. Int. Ed., 2001, 40, 417) recently reported the enzymatic formationof C—F bonds. These publications relate to a series of mutantglycosidase enzymes (wherein a conserved glutamate residue is replacedby various amino acids) which were able to generate glycosyl fluoridesby a deviant reaction, when the enzyme was incubated with fluoride at 2Mconcentration. The organo-fluorine product that was generated wasunstable (it was a glycosyl fluoride) and thus this process is notamenable to the synthesis of organo-fluorine compounds. Theconcentration of fluoride at 2 Molar is also very high indicating aninefficient process. These factors present poor prospects for commercialmanufacture.

The present invention provides a process for the synthesis of afluoronucleoside compound using a catalyst. Preferably the catalyst isan enzyme from Streptomyces cattleya. Preferably the enzyme is in a cellfree extract, and is at least partially purified.

The fluorinase enzyme has been purified to homogeneity and partialsequence data of the N-terminus of the enzyme is also presented (see SEQID No. 1).

Streptomyces cattleya is available publicly from the culture collectionheld by the Agricultural Research Service Culture Collection (NRRL) inUSA, under deposit No. NRRL 8057.

The present invention further provides an enzyme obtainable fromStreptomyces cattelya characterised in that said enzyme has the capacityto catalyse the synthesis of organo-fluorine compounds, particularlyfluoronucleosides. The enzyme is therefore a fluorinase enzyme. Theenzyme may be usefully presented in a cell-free extract, althoughpreferably the enzyme is at least partially purified. In someapplications the enzyme is purified to homogeneity. The preferredsubstrate of the synthesis reaction catalysed by the enzyme isS-adenosylmethionine (SAM). Optionally SAM is generated in situ by acombination of ATP or ADP and L-methionine, in the presence ofL-methionine S-adenosyl transferase. Organo-fluorine compounds producedby said enzymes include fluoroacetate and 5′-fluoro-5′-deoxyadenosine. Asuitable fluoride donor substrate of the synthesis reaction is afluoride salt, preferably a lithium, potassium or sodium fluoride salt,or is hydrogen fluoride.

The fluorinase enzyme has been purified to homogeneity. The variousstages of purification are shown in the SDS Page gel of FIG. 3. Themolecular weight of the fluorinase enzyme is 31.192 kd (confirmed byMALDI-TOF mass spectroscopy analysis). Non-denaturing gels indicate amolecular weight of 192 kd, suggesting that the active enzyme may be ahexamer. Edman degradation has indicated an N-terminal amino acidsequence as shown in FIG. 4.

In a further aspect, the present invention provides a fluorinase enzymeobtainable from Streptomyces cattleya having a molecular weight of 31kd.

The present invention further provides the use of an enzyme describedabove for fluoronucleoside synthesis.

As the enzyme originates from a Streptomyces genus and the molecularbiology of Streptomyces is very well developed, it is a very realprospect that in the future the gene for the enzyme could be cloned intoother organisms such that the engineered organism would then have animproved capacity to generate organo-fluorine compounds.

Promotors could be inserted into the fluorinase enzyme gene within thehost Streptomyces for similar effect.

Antibiotic biosynthesis genes from a range of Streptomyces sp. arecurrently being cloned to effect structurally modified antibiotics.There is a potential application to insert appropriate plasmidscontaining this gene into antibiotic gene clusters. The novel hybridclusters may generate fluorinated antibiotics by fermentation.

A suitable fluorination enzyme substrate for the synthesis reaction isS-adenosylmethionine (SAM). In a cell-free, partially purified orpurified, enzyme system SAM may be added as the substrate. However in acrude enzyme system (comprising ruptured cells from which cell debrishas been removed, suitably by centrifugation), ATP or ADP andL-methionine are sufficient to promote the fluorination. SAM may besynthesised in situ from ATP by the action of L-methionineS-adenosyltransferase. ADP disproportionate to AMP and ATP, followed bythe resultant conversion of ATP to SAM. The crude enzyme system hassufficient L-methionine S-adenosyltransferase present to catalyse theprocess.

One suitable substrate is a fluoride salt, preferably a lithium,potassium or sodium fluoride salt. Hydrogen fluoride is also suitable asa substrate. The concentration of the fluoride ions is preferably 2 to10 mM, although higher or lower concentrations of fluoride ions are alsopossible. Indeed significantly higher concentrations of fluoride ionsmay be of utility in certain commercial processes.

The organo-fluorine compound produced may be 5′-fluoro-5′-deoxyadenosine(5′-FDA). The 5′-fluoro-5′-deoxyadenosine may be used as an intermediateand in the production of other organo-fluorines, for example theorgano-fluorine compound fluoroacetate. In this case the crude enzymesystem will comprise additional enzymes, and will catalyse theconversion of 5′-fluoro-5′-deoxyadenosine to fluoroacetate as shown inScheme 1.

We have found that the more purified the enzyme system is, the greaterthe accumulation of 5′-fluoro-5′-deoxyadenosine as the biotransformationproduct.

The present invention further provides a coupled enzymebiotransformation process utilising the enzymes, L-methionineS-adenosyltransferase and the fluorination enzyme as catalysts andwherein L-methionine, ATP and fluoride are added as substrates.

A cell-free extract from the bacterium Streptomyces cattleya has theability to generate 5′-fluoro-5′-deoxyadenosine by the combination ofinorganic fluoride with ATP and L-methionine, or more preferably by thecombination of inorganic fluoride with S-adenosylmethionine, theco-factor generated by a combination of ATP and the amino acidL-methionine as shown in Scheme 2.

A crude extract from the bacterium Streptomyces cattleya will convertthe 5′-fluoro-5′-deoxyadenosine to fluoroacetate. A partially purifiedcell-free extract produces 5′-fluoro-5′-deoxyadenosine.

The process of the present invention generates a stable compound,containing a fluoromethyl group.

Nucleosides and their derivatives are widely used in DNA/RNA chemistryand as antiviral compounds. 5′-fluoro-5′-deoxyadenosine has applicationin a diverse range of potential applications in such research.

Preferably the organo-fluorine compound may be labelled with ¹⁸F forpositron emission tomography (PET). Preferably the source of ¹⁸F fromthe synchrotron is potassium fluoride, and preferably this is the samesource of fluoride used by the enzyme. Hydrogen fluoride may also beused and other fluoride salts are likewise possible. The half-life of¹⁸F is 110 minutes, and so rapid synthesis routes will be a considerableadvantage.

Fluorinated sugar (e.g. a fluoronucleoside compound) generated accordingto the present invention comprising ¹⁸F may be injected into a patient,and the patient may then be imaged.

Fluoroacetate has been used to study glial metabolism in the brain of arat and central nervous system of the rat. The invention provides asuitable route for the synthesis of ¹⁸F-fluoroacetate for positronemission tomography (PET) studies. This process introduces the fluorineenzymatically rather than chemically. The use of ¹⁸F-fluoroacetatesynthesised as described above for PET studies is thus part of thepresent invention.

[¹⁸F]-5′-fluoro-5′-deoxyadenosine may be used to explore the nucleicacid accumulation in tumour tissue.

PET offers the highest spatial and temporal resolution of all nuclearmedicine imaging modalities and can allow quantitation of tracerconcentrations in tissues. The use of ¹⁸F offers a number of advantagesover ¹¹C as a PET radionuclide, primarily because of its longerhalf-life (110 minutes for ¹⁸F versus 20 minutes for ¹¹C). From aradiochemistry and radiopharmacy perspective, ¹⁸F-labelledpharmaceuticals allows substantially more time for radiochemicalsynthesis, purification and quality control of doses for use in vivoexperiments.

¹⁸F-labelled radiopharmaceuticals can be synthesised according to theprocess of the present invention in quantities sufficient for theformulation of multiple doses from a single production and for remotedistribution to locations without on-site cyclotron facilities.Aliphatic ¹⁸F labelled compounds are commonly produced by nucleophilicsubstitution with [¹⁸F]F⁻ for a nucleofuge (i.e. halogens, sulfonates,cyclic sulfamidates) in a polar aprotic solvent (i.e. acetonitrile,dimethylsufoxide) and therefore only a limited set of synthetic methodsare available for rapid radiofluorination. The radiolabelling of[¹⁸F]-5′-fluoro-5′-deoxyadenosine by nucleophilic substitution using5′-halogenated adenosine derivatives as precursor fails to produce thedesired product with good yield (Lethel Sz., Horváth G., Boros I.,Mikecz P., Trón L. Journal of Radioanalytical and Nuclear Chemistry, vol245, No 2 (2000) 399-401). The method of the present invention providesa means of radiolabelling PET isotopes leading to novelradiopharmaceuticals or improved availability of existing tracers.

The process of the present invention provides a process forenzymatically synthesising radiolabelled[¹⁸F]-5′-fluoro-5′-deoxyadenosine ([¹⁸F]-5′-FDA) for PET.

It is a very real prospect that the biosynthesis of5′-fluoro-5′-deoxyadenosine by the present invention will enable a novelpreparation of 5-fluoro-5-deoxyribose by either further chemical orenzymatic treatment of the enzymatic product5′-fluoro-5′-deoxyadenosine, such that the adenine base ishydrolytically cleaved.

The invention will now be described by way of example only, with respectto the figures in which:

FIG. 1—shows a HPLC chromatogram of reaction mixture aliquots; 5 to 20%MeCN in 50 mM KH₂PO₄; flow 1 ml/min.

FIG. 2—shows a HPLC chromatogram of [¹⁸F]5′-FDA co-injected withstandard 5′-FDA C18 column; 5 to 20% MeCN in 50 mM KH₂PO₄; flow 1ml/min.

FIG. 3—shows the purification protocol for the fluorine enzyme whereinLane 1 shows molecular weight markers, Lane 2 shows crude cell freeextract, Lane 3 shows 45-60% (NH₄)₂SO₄ cut, Lane 4 shows phenylsepharose column, Lane 5 shows gel filtration superdex 200, Lane 6 showsanion exchange Source 15 Q. The Lanes are numbered from left to right.

FIG. 4—shows the N-terminal 25 amino acid sequence of the fluorinaseenzyme.

EXAMPLE 1

Experimental Procedures

Materials and Culture Conditions

Cells of S. cattleya (NRRL 8057) were grown under conditions asdescribed in K. A. Reid, J. T. G. Hamilton, R. D. Bowden, D. O'Hagan, L.Dasaradhi, M. R. Amin and D. B. Harper, Microbiol., 1995, 141, 1385.After 7 days of incubation the cells were harvested by centrifugation(9000 rpm, 25 min), washed three times with Tris buffer (100 mM, pH 7.8)and stored at −20° C. until required. ATP was purchased from Fluka,other biochemicals were purchased from Sigma. Sonication was carried outwith a vibra cell (Sonic & Materials), ¹⁹F and ¹H-NMR were recorded on aVarian Inova NMR spectrometer. Enzyme purification was performed with anAKTA Prime System (Pharmacia). For Analytical and semipreparative HPLC aVarian System was used, containing a pump (9012, Varian) and variablewavelength UV detector (9050, Varian).

Enzylmatic Preparation of 5-fluoroadenosine in a Cell Free Extract ofStreptomyces cattleya

A cell-free extract of S. cattleya was prepared from frozen cells whichwere resuspended in Tris-buffer (100 nM, pH 7.8) containing potassiumbicarbonate (200 mM) (0.2 g wt cells weight/ml). The cell suspension wassonicated (three pulses of 30 sec duration at 40-50% microtip power) andthe cell debris was removed by centrifugation (20,000 rpm, 20 min). Tosupernant (900 μl) was added ATP (5 mM), MgCl₂ (15 nM), L-methionine(0.2 mM), potassium fluoride (10 mM) in a total volume of 1 ml andincubated for 16 hours at 26° C. in a water bath. ¹⁹F-NMR analysisdemonstrate the formation of an organofluorine compound δ_(F) (470.445MHz; ²H₂O)-229.65 (J 47.5, 30.1). Incubation of cell-free extract (970μl) supplemented with SAM (0.4 mM) and potassium fluoride (10 mM) for 16hours at 26° C. also resulted in the formation of5′-fluoro-5′-deoxyadenosine.

Preparation of 5′-fluoro-5′-deoxyadenosine Using a Purified ProteinExtract from Streptomyces cattleya

A cell-free extract from S. cattleya cells was prepared as describedabove. Ammonium sulfate was added to the cell-free extract and theprecipitate at 45-60% saturation was kept. The precipitate wasresuspended in Tris-buffer (100 mM, pH 7.8) containing potassiumbicarbonate (200 mM) and desalted using a HiTrap desalting column (5 mlbed volume, Pharmacia). The protein was then applied to an anionexchange column (HiTrap QXL, 1 ml bedvolume, Pharmacia) previouslyequilibrated with the same buffer. The column was washed with 20 ml ofbuffer followed by 20 ml of Tris-buffer (100 mM, pH 7.8) containingpotassium bicarbonate (200 mM) and KCl (1 M). The collected fractionswere assayed by incubating a small volume (900 μl, 970 μl) with eitherATP (5 mM), MgCl₂ (15 mM), L-methionine (0.2 mM), potassium fluoride (10mM) or with SAM (0.4 mM) and potassium fluoride (10 mM) for 16 hours at26° C. Only in the presence of SAM and fluoride was formation of5′-fluoro-5′-deoxyadenosine observed.

Purification of 5′-fluoro-5′-deoxyadenosine

For purification of the organofluorine product, a cell-free extract (15ml) was prepared as described before and incubated with ATP (5 mM),MgCl₂ (15 mM), L-methionine (0.2 mM), potassium fluoride (10 mM) for oneday at 26° C. After that time the biotransformation mixture was heatedat 90° C. for 10 minutes and denatured protein was removed bycentrifugation. The supernatant was lyophilised the resultant powder wasdissolved in H₂O (2 ml) and remaining solids were removed bycentrifugation. The clear supernatant (100 μL) was applied to a Hypersil5 μm C-18 column (250×10 mm, Phenomenex) and eluted isocratically(potassium phosphate buffer (50 mM)/acetonitrile, 95.5) at a flow rateof 5 ml/min. Detection was by UV at 260 nm.

Fractions corresponding to observed peaks were collected and analysed by¹⁹F-NMR. Fractions containing the fluorinated product were combined,lyophilised and dimethyl-d₆ sulfoxide (1.5 ml) was added. The solidswere removed by centrifugation and the clear supernatant was used forstructure elucidation by ¹H and ¹⁹F-NMR. δ_(H) (500 MHz; DMSO) 4.11(2×m, J_(FH) 21.4, 1H, 4′-H), 4.20 (t, J5.0, 1H, 3′-H), 4.49 (t, J5.0,1H, 4′-H), 4.62 (2×m, J_(FH) 47.8, 2H, 5′-H), 5.90 (t, J5.0, 1H, 1′-H),8.06 (s, 1H, 2-H), 8.20 (s, 1H, 8-H); δ_(F) (470.553 MHz; DMSO)-229.69(J 48.9, 30.1).

EXAMPLE 2

The partially purified fluorinase enzyme (0.4 mg/ml protein) wasincubated with [¹⁸F]HF (in solution in [¹⁸O]H₂O) and SAM (0.4 mM) at 23°C. at pH=7.0. The aliquots collected at different time points werepassed through an anion exchange column and injected into ahigh-pressure liquid chromatography (HPLC) system coupled to aradioactivity detector to follow the course of the radiolabelling.

Analysis of the aliquots by radio-HPLC showed two radioactive peaks. Theretention time of the first radioactive signal eluting at 3.6 minutes onthe reverse-phase column (corresponding to the dead volume of thecolumn) is consistent with unreacted [¹⁸F]fluoride or polar productssuch as [¹⁸F]fluorine bound to proteins. The only radioactive non-polarpeak was detected at 15.6 minutes (FIG. 1). When aliquots wereco-injected with an authentic sample of 5′-FDA, the UV signal of thereference compounds co-eluted with the radioactive peak eluting at 15.6minutes (FIG. 2). The apparent difference in the retention times(approximately 0.5 minutes) is an artefact to physical separation of theradioactivity and UV detectors (FIGS. 1 and 2).

In conclusion, an enzymatic radiolabelling method for the production of[¹⁸F]-5′-FDA has been successfully developed starting from SAM and[¹⁸F]HF.

1. A process for the synthesis of a fluoronucleoside compound, said process comprising mixing a substrate, a fluoride compound and an enzyme comprising an amino acid sequence SEQ ID NO:1 as a catalyst, said enzyme obtainable from Streptomyces cattleya.
 2. A process as claimed in claim 1 wherein the enzyme is at least partially purified.
 3. A process as claimed in claim 1 wherein the substrate comprises S-adenosylmethionine.
 4. A process as claimed in claim 1 wherein said fluoronucleoside compound is synthesised by mixing a fluoride salt with a crude enzyme system from Streptomyces cattleya comprising ATP and L-methionine or ADP and L-methionine together with L-methionine S-adenosyltransferase.
 5. A process as claimed in claim 1 wherein the fluoride compound is a lithium, potassium or sodium fluoride salt.
 6. A process as claimed in claim 1 wherein said fluoride compound is hydrogen fluoride.
 7. A process as claimed in either claim 5 wherein the concentration of fluoride ions is 2 to 10 mM.
 8. A process as claimed in claim 1 wherein the fluoronucleoside compound is 5′-fluoro-5′-deoxyadenosine.
 9. A process as claimed in claim 1 wherein the fluoride compound is labelled with ¹⁸F.
 10. A process as claimed in claim 9 wherein the fluoronucleoside compound is labelled with ¹⁸F.
 11. A process as claimed in claim 1 wherein the enzyme is obtained from Streptomyces cattleya. 